Ability of Trichoderma harzianum from Semi Arid Soils to Enhance Antioxidant Defense of Maize Seedlings under Water Stress

Aim: Determine the effect of different inoculum concentrations of T. harzianum from semi - arid soils on the activity of antioxidant enzymes of maize seedlings under water stress. Methods and Results: This study employed a three-factor factorial (3×4×4) design, arranged in a completely randomized design (CRD) with three replications. Three maize varieties (H614, H629 and H6210) were treated with four concentrations of T. harzianum (0, 1x10 5 , 1x10 7 and 1x10 10 spore/ml and thereafter grown under four osmotic potential regimes (0, -0.3, -0.6 and -0.9 MPa). Results from the study showed that T. harzianum had a significant effect on Superoxide dismutase (SOD) and catalase (CAT) activity of maize seedlings and did not enhance either maize seed germination or seedling growth. The activity of SOD and CAT was significantly enhanced by T. harzianum in all the three varieties of maize. Optimum SOD and CAT activity were recorded in seeds treated with 10 7 spores/ml of T. harzianum . Under normal growth conditions (0MPa), SOD and CAT activities were not enhanced by T. harzianum . However, under severe water stress (-0.9MPa), maximum activity of the enzymes was registered in all the three varieties of maize. Conclusion: Maize seedling colonization by T. harzianum enhanced systems of antioxidative enzymes. Maximum activity of these enzymes was recorded under severe water stress (0.9MPa) mainly in seedlings treated with 10 7 spores/ml of T. harzianum . Consequently T. harzianum from semi-arid soils may be employed to improve maize plants’ tolerance to water stress. Significance and Impact: With rapid increase in human population, coupled with global climate change, there is need to devise a cheap and safe option to increase the production of food crops. The ability of T. harzianum in promoting plant growth precisely maize under stress is of importance.


INTRODUCTION
Water stress or drought stress is an inevitable and recurring feature in global agriculture. It is one of the most devastating environmental stresses. Water stress limits growth and productivity of main crop species, reducing yields to less than half [1]. Also it has been reported that, about one-third of the world's potentially arable land suffers from water shortage [2]. Maize (Zea mays L.) also known as queen of cereals is an important cereal crop grown all over the world [3] and is central to developing nations' agriculture and food security. Most cereals, maize being one of them are drought-sensitive. Significant yield losses can occur in even a mild water stress during reproductive phase [4]. Water stress brings about physiological, biochemical and molecular changes in plants which oversees growth and productivity. One such biochemical mechanism includes antioxidant enzymatic system (superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) etc.), which protect plant cells against the detrimental effects of reactive oxygen species (ROS) generated under variety of environmental stresses [5]. [6] showed that Trichoderma spp. are cosmopolitan fungi found in agricultural, forest, desert soils. They also colonize roots of various plants found in different ecosystems including maize. They have been defined as plant symbiont opportunistic avirulent organisms, able to colonize plant roots and to produce compounds that stimulate growth and plant defense mechanisms under suboptimal conditions. Trichoderma spp. are the most common research tools as microbial inoculants which have been mostly used as biocontrol agents. However, in the recent years, they have become popular as plant growth promoters [7]. For Trichoderma to effectively augment plant development, it must be able to establish in the spermosphere of germinating seeds, distribute on the emerging radicle and colonize the developing root [8]. Research shows that colonization of host roots with Trichoderma strains enhances entire tolerance to biotic and abiotic stresses [9,10]. Such kind of augmented tolerance to biotic and abiotic stress is believed to be due to enhanced root growth and the nutritional status of plants [11].
An increase in damaging levels of reactive oxygen species (ROS) is a common feature in plants in the presence of abiotic stresses [12]. Even though, the ability of Trichoderma spp. mainly T22-to alleviate varied types of stress is suggested to be mediated by enhanced redox buffering capacity of the colonized plants. For example, under water deficit, lipid peroxide content of colonized tomato seedlings was lower than in the control seedlings [13]. Lipid peroxidation is commonly associated with oxidative damage [14] when the level of ROS exceeds the capacity of the antioxidant defense system [12,15]. While changes in ROS level may act as a signal to activate a host of defense mechanisms, continued production of high levels of ROS under ongoing stress causes damage to plants [12].
Superoxide dismutase is the main scavenger of superoxide radicals, which converts the toxic superoxide (O 2 -) to hydrogen peroxide and oxygen, through a process called dismutation reaction: 2O 2¯ + 2H+ The enzyme embodies the first line of cell defence against ROS generated abiotic stresses like drought in plants, therefore, preventing the tissue damage due to oxidative stress. CAT and POD enzymes are able to convert toxic H 2 O 2 to water and oxygen. under water stress only elevated SOD activity cannot protect the plants from toxic effect of oxygen free radical hence CAT and POD is needed to remove toxicity of H 2 O 2 [16]. On the other hand, Trichoderma strains have been reported to enhance the activity of these pathways, through improved expression of genes encoding the component enzymes [13]. For example, if these pathways are enhanced in the chloroplasts, then it is expected that the photosynthetic efficiency will increase by reducing damage by the superoxide anion and other reactive species involved in photosynthesis [17]. Trichoderma spp. augments protection against ROS perhaps by increasing ROS scavenging capacities. Proteomics of roots inoculated with Trichoderma showed an increase in levels of anti-oxidative enzymes mainly Superoxide dismutase (SOD) as well as increased levels of peroxidase, glutathionereductase and Glutathione-S-transferase (GST), and other detoxifying enzymes in leaves [18]. In a recent experiment performed by [19], T. harzianium (T-35) benefited rice plants by increasing their tolerance to severe drought stress through the reduction of oxidative stress by enhancing the production of SOD, CAT and POD anti-oxidative enzymes. Furthermore, studies have shown that microbes in harsh habitats are adapted to their environments and they have the ability to transfer this ability to their host plants grown in such habitats like arid and semi arid area [20]. Therefore, the main goal of this study is to investigate the ability of T. harzianum isolated from semi-arid soils to enhance the antioxidant defense in maize seedlings.

Soil Sample Collection
Soil samples were collected from the semi-arid rangeland of Marigat area, Baringo County Kenya. The area is located between latitude 00 o 26-00 o 32'N and longitude 36 o 00'36 o 09' E. The climate is semi-arid with an average altitude of 900 M above the sea level. A total of 60 g of soil samples were randomly collected from the rhizosphere of grass plants and bare soil in 10 cm depth using a sterile soil auger. The samples were then transferred into sterile polyethylene bags and transported to the Microbiology laboratory, at the University of Eldoret, Kenya within 24 hours of collection. These samples were used for isolation of T. harzianum. [21] method for T. harzianum isolation was adopted with slight modifications. Ten grams of the soil sample made up to 1000 ml using sterile distilled water in a sterile conical flask. The soil suspension was left for one hour at room temperature to release conidia and hyphae adhering to soil particles. Serial dilutions up to 10 -3 were prepared from the suspension and 1 ml aliquots were then spread-plated onto Potato Dextrose agar (PDA) medium supplemented with 50 mg/l of streptomycin antibiotic to inhibit bacterial growth. The plates were then incubated at 28°C and 35°C for seven days. Under 35°C, growth inhibition of all species of Trichoderma has been reported except for T. harzianum [22]. Distinct colonies of T. harzianum were picked based on their on their morphological characteristics as described by [23]. To obtain pure cultures of T. harzianum, streaking was done on fresh PDA medium twice. Microscopic examination and measurements of conidiophores and conidia were made from slide preparations stained with lactophenol-cotton blue and observed under a light microscope under ×400. Pure cultures of T. harzianum were then taken to Kenya Agricultural and livestock Research Organization (KALRO) Njoro, Kenya for confirmation.

Inoculum Production of Trichoderma spp
The study adopted [24] method for production of T. harzianum inoculum. However, slight modification was made to suit the present study. The pure cultures obtained above were subcultured aseptically in eight 90 mm diameter Petri plates each containing 15 ml of a freshly autoclaved PDA media. Incubation of the eight plates was done at 28°C for ten days. On the tenth day, spore suspensions from the fungus inoculum were prepared by flooding the surface of the agar slant with 10 ml sterile distilled water and the culture surface gently scraped to extricate the spores. The spore suspensions derived from the eight Petri plates were transferred separately to 500 ml flasks containing 400 ml sterile distilled water. Flasks were then shaken for 2 minutes to ensure that the spores are appropriately mixed. Four concentrations of the fungal spore (0, 1x10 5 , 1x10 7 and 1x10 10 spore/mL) were determined using a haemocytometer under a light microscope at ×400 magnifications. The control was made up of autoclaved spores of T. harzianum. The autoclaving process was done at 121°C for 15 minutes [25].

Water Stress Induction
Polyethylene glycol 6000 (PEG) at different concentrations was prepared to establish different levels of osmotic potential. Approximately 0, 143.18, 213.64 and 267.97 g of PEG were dissolved in 1000 ml distilled water to generate four osmotic stress levels (0, -0.3, -0.6 and -0.9 MPa, respectively).The control was made up of only distilled water with no PEG.

Research Design
The study employed a three-factor factorial design (3×4×4) replicated three times. Maize seeds belonging to (H614, H629 and H6210) varieties with no cracks or any visible deformations were used in this study. Surface sterilization was done for 5 minutes with 1% sodium hypochlorite solution, followed by rinsing with distilled water three times and finally air dried. Wet seed treatment method was adopted, where seed coating was done by applying 2% of starch (adhesive) on the maize seeds. Subsequently, maize seeds were dipped in seed coating suspension of 0, 1×10 5 , 1x10 7 and 1×10 10 spores/mL Trichoderma harzianum for 2 minutes. The seeds were finally germinated on petri dishes lined with whatman filter paper satured with distilled water under 0, -0.3, -0.6 and -0.9 MPa for ten days.

Enzyme Extraction from Plant Samples
Extraction of (SOD and CAT) enzymes from plant samples was done according to [26]. Both water stressed and control maize seedlings were evaluated for antioxidative enzymes' activity after 10 days of germination. Fresh weight of 0.5 g leaf sample was taken and then placed in a freezer at -10°C for 24 hrs. The frozen leaf sample was then finely ground by pestle in a frozen motor to prevent the loss of enzymes' activities. The frozen powder was added to 10 mL of phosphate buffer (pH 7.5). The homogenate was centrifuged at 15000 × g for 10 min at 25°C and supernatant was used as enzyme source for catalase (CAT) and superoxide dismutase (SOD).

Statistical Analysis
The experiment for the activity of SOD and CAT enzymes was carried out using (4×3×3) factorial design with three replicates. The mean values (±SE) of SOD and CAT enzymes activity of the three replicates were calculated. The mean values were then analyzed by a three-way analysis of variance (ANOVA) using Statgraphics programme to determine the activity of antioxidant enzymes (SOD and CAT). The means were separated using Tukey's test.

Isolation of T. harzianum
At 28°C and 35°C, T. harzianum grew uniformly and formed white mycelia within five days.
After ten days of growth, the fungus displayed green conidia, at both 28 and 35°C. The conidia production was dense at the center and towards the margins. It was also observed that, conidia production by the fungus was not different at 28 and 35°C as shown in Plate 1. Table 1 showed that concentration of T. harzianum and osmotic potential affected SOD and CAT activities significantly (p<0.05).
Concentration of T. harzianum by osmotic potential and maize variety by osmotic potential interactions were also significant (p<0.05) for SOD and CAT activities. However, maize variety, interactions for maize variety by T. harzianum concentration and maize variety by T. harzianum concentration by osmotic potential had no significant (p>0.05) effect on SOD and CAT activities. At low osmotic potential, increase in concentration of the fungus increased CAT and SOD activity until 10 7 spores/ml of the fungus. Further increase in concentration (10 10 spores/ml of T. harzianum) led to a stabilization of the activity.
Results showed that SOD activity increased significantly (p<0.05) with decrease in osmotic potential in both treated and untreated maize seedling across the three varieties of maize ( Table 2). Also SOD activity increased with increase in concentration of T. harzianum upto 10 7 spores/ml of the fungus before stabilizing with 10 10 spore/ml of the fungus. Maize varieties did not differ significantly in SOD activity at the same osmotic potentials with the same spore concentration of T. harzianum. Under normal growth condition (0 MPa), SOD activity increased significantly (p<0.05) from 15.0 U g -1 protein in control to 15.2 U g -1 protein in seedlings treated with 10 5 spores/ml of T. harzianum. Further increase (15.5 U g -1 protein) in SOD activity was recorded in seedlings treated with 10 7 spores/ml of T. harzianum. Stabilization of SOD activity (15.5 U g -1 protein) was recorded in control seedlings treated with 10 10 spores/l of the fungus for the three varieties of maize as shown in Table 2.
Under severe water stress (-0.9 MPa), SOD activity increased significantly (p<0.05) from 194 U g -1 protein in control to 337 U g -1 protein in seedlings treated with 10 5 spores/ml of T. harzianum. Maximum SOD activity (893 U g -1 protein) was recorded in seedlings treated with 10 7 spores/ml of T. harzianum with stabilization of SOD activity (892U g -1 protein) recorded in seedlings treated with 10 10 spores/ml of the fungus across the three varieties of maize ( Table 2).
Seedlings treated with 10 7 and 10 10 spores/ml concentrations of T. harzianum were not significantly different (p>0.05) in SOD activity in all the three varieties of maize. However, they were significantly different (p<0.05) from seeds treated with 10 5 spores/ml of T. harzianum and control (Table 2). Furthermore, seedlings treated with 10 5 spores/ml of T. harzianum showed significant (p<0.05) SOD activity from control irrespective of maize variety.  Similarly, CAT activity increased significantly (p<0.05) with decrease in osmotic potential in both treated and untreated maize seedling across the three varieties of maize (Table 2). CAT activity increased with increase in concentration of T. harzianum upto 10 7 spores/ml of the fungus before stabilizing with 10 10 spore/ml of the fungus. Maize varieties did not differ significantly in CAT activity at the same osmotic potentials with the same spore concentration of T. harzianum.
CAT activity increased significantly (p<0.05) from 0.01µmol H 2 O 2 min-1 g -1 protein in control to 0.06 µmol H 2 O 2 min-1 g -1 protein in control seedlings treated with 10 5 spores/ml of T. harzianum. Seedlings treated with 10 7 spores/ml of T. harzianum recorded highest CAT activity (0.09 µmol H 2 O 2 min-1 g -1 protein), while stabilization of CAT activity (0.09 µmol H 2 O 2 min-1 g -1 protein) was recorded in seedlings treated with 10 10 spores/ml of the fungus at 0 MPa a cross the three varieties of maize as shown in Table 3.
At -0.9 MPa, CAT activity increased significantly (p<0.05) from 1.0 µmol H 2 O 2 min-1 g -1 protein in control to 1.3 µmol H 2 O 2 min-1 g -1 protein in seedlings treated with 10 5 spores/ml of T. harzianum. Maximum CAT activity (4.0 µmol H 2 O 2 min-1 g -1 protein) was recorded in seedlings treated with 10 7 spores/ml of T. harzianum. Stabilization of CAT activity (4.0 µmol H 2 O 2 min-1 g -1 protein) was recorded in control seedlings treated with 10 10 spores/ml of the fungus for the three varieties of maize as shown in Table 3.

DISCUSSION
In this study, we isolated T. harzianum from semi-arid soils. There was no doubt that the isolated fungus was T. harzianum since growth at 35°C was recorded. [22] found that the capability of T. harzianum to grow at 35°C was useful in distinguishing it from other Trichoderma species.
Findings from the present study clearly showed that T. harzianum played a key role in enhancing maize seed germination and early seedling growth under water stress. Seeds treated with T. harzianum showed significant difference in germination from control at water stress. Seeds respond to T. harzianum very early in germination, even before the radicle protrudes [6]. Also, Trichoderma spp. have been shown to augment seed germination by enhancing phase III imbibition (cell elongation, followed by radicle protrusion). The present results are in agreement with those of [13]. The authors found that tomato seeds that were treated with T. harzianum (T22) showed higher seed germination percentage than untreated tomato seeds.  levels (0, -0.3, -0.6 and -0.9 [18]. MeJA induces expression of genes encoding antioxidant enzymes. MeJA may play a signaling role in the expression of genes encoding antioxidant enzymes as well. Similar findings were reported by [10] when proteomics of shoots inoculated with Trichoderma showed an increase in levels of anti-oxidative enzymes mainly Superoxide dismutase as well as increased levels of peroxidase, glutathione-reductase and Glutathione-S-transferase (GST), and other detoxifying enzymes in leaves.
In the present study, T. harzianum increased SOD and CAT activities significantly in all the three varieties of maize under water stress as compared to control plants. These results are in agreement with those of [31] where a transient increase in intracellular ROS was detected 5 to 10 min after treating soybean cell culture with culture filtrate of T. atroviride. Furthermore, [32] also reported that T. harzianum enhanced the activity of antioxidant enzymes in tomato plant subjected to water stress.
SOD converts superoxide radicals to hydrogen peroxide, while CAT enzyme converts hydrogen peroxide to water and oxygen [30]. Increased activity of SOD alone cannot protect plants from toxic effect of oxygen free radicals and therefore, other enzymes like CAT and POD are required to get rid of hydrogen peroxide toxicity [16].

CONCLUSION
This study presents evidence that maize seedling colonization by T. harzianum enhances systems of antioxidative enzymes. Maximum activity of these enzymes was recorded under severe water stress (-0.9 MPa) mainly in seedlings treated with 10 7 spores/ml of T. harzianum. This consequently indicates that, one of the mechanisms that T. harzianum especially those isolated from semi-arid soils employ in improving plant tolerance to water stress is through the reduction of oxidative stress via increased. SOD and CAT activities. Treatment of seed or plants that could simultaneously confer resistance to abiotic stresses would be of importance to agricultural plant production.